Novocrania in situ en wnt1 wnt5 2nd try 

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Second chance on the Novocrania in situ which failed last time. This time I’ll do 7 minutes of ProtK and wash extensively with glycine to stop the reaction.

3 embryos of each stage: radial, bilateral, early larva, and larva.

engrailed wnt1 wnt5
  • Initially in MeOH.
  • 1x 5min 60% MeOH 40% PTw.
  • 1x 5 min 30% MeOH 70% PTw.
  • 4x 5 min PTw.
  • ProteinaseK for 7 min.
  • Stopped exactly at 7 min with 900 µL of glycine.
  • 1x 5 min glycine.
  • 1x 5 min 1% TEA.
  • 1x 5 min 1% TEA + Ac. An. –> embryos stick a bit to bottom.
  • Added 100 µL of Ac An. embryos continue to stick.
  • Washed 2x PTw 900 µL.
  • Fixed in 4% FA for 1h25.
  • Washed 3x PTw.
  • 1x 10 min PTw at 80 °C.
  • 1x 5 min PTw.
  • 1x 10 min Hybe Buffer at RT.
  • Exchanged hybe and incubate at 62 °C.


  • Added probes.
  • Embryos were still looking ok, although somewhat transparent.


  • Larvae look damaged and may fall apart. Saw a bilateral missing a piece… Otherwise it looks better than last time. I think the time or concentration of ProtK (newly opened) really needs to be decreased!
  • 10 min and 40 min washes with hybe wash.
  • Series of 30 min hybe to 2x SSC.
  • 3x 20 min 0.2X SSC + 0.1% Tween-20.
  • Grade-series of 0.2X SSC to PTw 10 min each.
  • 5x 5 min PBT.
  • Incubate 1h with 1x blocking buffer made with the last 500 µL of 5x blocking buffer from the fridge and newly made maleic acid buffer.
  • Exchanged for 1:5000 Anti-DIG-AP in blocking buffer and incubated overnight in cold room.


Embryos still look integer although larvae are damaged.

  • Started washing with PBT at 13h.


  • Began to develop at 16h.
  • Faint band of engrailed at 19h.
  • Noticed some precipitated when exchanging NBT/BCIP, maybe old BCIP (it is running out)?
  • Let overnight in the cold room.


  • Checked at 11h and the engrailed stripe was more prominent; nothing yet in the others.
  • There are crystals forming in the bottom :( and some clear precipitate floating around…
  • I exchanged the AP substrate and let it go under RT.
  • Exchanged again after 2h.
  • Final exchange and cold room at 17h30. Maybe there was a bit of background coming up in engrailed… not sure so I let it overnight and will stop tomorrow.


  • engrailed well began to show some background and the others were looking bad with crystals packing up. So I stopped them all.
  • AP -magnesium 2x.
  • Ethanol washes.
  • 70% Glycerol exchanged once for glycerol with 1:5000 DAPI.
  • I restarted the wnts because I felt bad. Let them develop overnight.


  • Stopped wnts and did alcohol series.
  • Mounted and took photos of engrailed.